The proposed research concerns the generation of oxygen metabolites that are of vital importance in the killing of microbes and other invasive cells and organisms by neutrophils and related phagocytic cells. Patients whose neutrophils have an impaired capacity for the respiratory burst suffer from chronic infections and usually die in childhood. It has long been evident that internalization of microbes, or even inert latex beads, by neutrophils, a process termed phagocytosis, is accompanied by a transient respiratory burst and production of lethal oxygen metabolites like superoxide anion and hydrogen peroxide. Oxygen consumption may increase 20 x during phagocytosis. The respiratory burst is known to require pyridine nucleotides and an NADPH dehydrogenase. A novel cytochrome b has also been implicated. The proposal is to study (a) whether an electron transport chain requiring ubiquinone is necessary in neutrophils and other phagocytic cells for production of the lethal oxygen metabolites and to study (b) whether neutrophils are activated when separate segments of the electron transport chain are brought together during the membrane fusion that accompanies phagocytosis. The experimental objectives are (1) to isolate from fresh human blood and rat peritoneal cavities highly enriched preparations of neutrophils, eosinophils and macrophages/monocytes using gradient techniques and to determine the ubiquinone content and function in each, (2) to fractionate, using rate zonal centrifugation techniques, resting and activated neutrophils and to compare the distribution patterns of electron transport components, (3) to resolve ubiquinone from activated neutrophils by careful extraction with pentane and to test by reconstitution whether ubiquinone is required for the respiratory burst, and (4) to purify individual protein components and to show by reconstitution which are necessary and regulatory for microbicidal events.